Dedicated to Mario Autuori (in memoriam) and Walter Hugo de Andrade Cunha who greatly contributed to the leaf-cutting ant studies. We acknowledge the support of S&#227;o Paulo State University and the Institute of Biosciences. This study was in part financed by the Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior-Brasil (CAPES) - Finance Code 001, Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (CNPq), Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de S&#227;o Paulo (FAPESP), and Funda&#231;&#227;o para o Desenvolvimento da UNESP (Fundunesp).

The authors have no conflicts of interest to disclose.

The protocol described here to maintain leaf-cutting ant colonies has been developed and applied for over three decades in an assertive and replicable way. It allowed the development of research that would be limited by field conditions. Thereby, healthy ants and colonies became available for research in several areas such as comparative morphology, toxicology51,52, histology53, and microbiology54,<sup cl...

Ants compose a diverse group of individuals that exert an influence on most terrestrial environments1. They act as efficient dispersers2,3,4, predators5 and ecosystem engineers6,7,8,9,10, highlighting their importance and ecological success on natural ecosystems. All ant species are classified as eusocial insects; however, their social organization varies greatly among different species groups, i.e., labor division systems, functional groups, communication among individuals, forage organization, colony foundation, and reproduction process11. As a highly diversified group, they resort to several food resources and specialized feeding behaviors. As a matter of fact, agriculture was not only a huge step for human civilization, but also for ant species. Approximately 55 to 65 Ma ago12, attine ants began to culture fungi and incorporate them into an almost exclusive diet. They became so specialized that they developed strict, dependent, and obligatory interactions classified as symbiosis, where one individual does not survive without the other.
Lower fungus-growing ants collect and process dead organic matter, such as fragments of rotting leaf, to grow their mutualistic fungus; while higher fungus-growing ants harvest fresh plant material, composing one of the most successful symbiotic natural systems13. This highly specialized agriculture technique allowed them to seize a new niche. The higher attine ants comprise the leaf-cutting ants, a monophyletic group that arouse between 19 Ma (15-24 Ma) and 18 Ma (14-22 Ma)14,15,16 consisting of four valid genera: Atta Fabricius, Acromyrmex Mayr, Amoimyrmex Cristiano, and Pseudoatta Gallardo. The leaf-cutter agriculture system performed by the leaf-cutting ants, evolved from derived agriculture systems17. Most of these species exclusively exploit the mutualistic fungus species Leucoagaricus gongylophorus Singer18 (also called Leucocoprinus gongylophorus Heim19), marking a significant evolutionary transition11. The fungal cultivars are transmitted vertically, from original nests to offsprings, suggesting that they are clonally propagated20.
Remarkably, Atta societies developed a complex organizational structure of enormous importance in their environment and of great interest to myrmecologists. Their population can be composed of millions of individuals, most of them sterile female workers that display an accentuated polymorphism, i.e., distinct size and anatomical morphology. The population is distinguished by castes according to age, physiological state, morphological type, behaviors, and specialized activities in the colony21. Workers can be discriminated into gardeners and nurses, within-nest generalists, foragers and excavators, and defenders or soldiers21. This organization allows the performance of tasks in cooperation and a self-organizing system that can produce highly structured collective behaviors, allowing them to respond efficiently to environmental disturbances22.
The role of population renewal is played by a single queen (i.e., monogynous), for as long as she lives, constituting the permanent reproductive caste22. Atta queens are known to live for more than 20 years, laying eggs throughout their lifespan23. As the queen is irreplaceable, its endurance is crucial for the survival of the colony13,20,23,24. Yet, thousands of winged reproductive females and males can be found in the nest during breeding seasons, but none stays in the original nest, forming a temporary caste22. In Atta sexdens colonies, nearly 3,000 reproductive females and 14,000 reproductive males are produced25. It occurs when a colony reaches sexual maturity, approximately 38 months from its implementation, and is repeated annually ever since until it is extinguished23,25. New Atta colonies are established through haplometrosis, where one queen commences a new nest.
When environmental conditions are favorable, the reproducers leave the underground nest to begin the nuptial flight. The period of its occurrence differs by region, ranging along the year throughout the brazilian territory depending on the species. However, the event seems to be preceded by rainfalls and humidity elevation26, which can be related to excavation facilitation due to soil moisture22. Frequently, 1-5 weeks before the nuptial flight, nest entrances and channels are widened to facilitate the reproductive individuals to depart. Before leaving their mother colonies, the winged females collect and store, in an infrabuccal cavity, a portion of the mutualistic fungus20,27. Multiple copulations are performed mid-flight, and it is calculated that one queen can be inseminated by three to eight males (i.e., polyandry) in some species28, ensuring genetic variability29. Afterward, the queens proceed to the soil, giving preference to locations with no or few vegetation25, where they remove their wings and excavate their first nest chamber. This is the only period where queens can be seen outside the nest. Although individuals of the temporary caste were seen in artificial nests, it is unknown whether any successful copulation (i.e., nuptial flight) was performed in laboratory conditions24.
The initial nest construction corresponds to the most crucial period of the colony, which can last from 6 h to 8 h23,25. At this moment, the queen cloisters herself in the initial chamber, and in a matter of days, oviposition begins. The first eggs are fed to the mycelial that the queen regurgitates, marking the start of the colony&#39;s fungus garden. The first larvae appear in approximately 25 days22, and nearly at the end of the first month, the colony consists of a mat of proliferating fungus, where immatures (eggs, larvae, and pupae) are nested, and the queen, who raises her initial offspring in isolation23. Eggs are also the food resource of the first larvae and highly consumed by the queen13. Additionally, the queen sustains herself with fat-body reserves and catabolizing wing muscles that are no longer of use13. The initial fungus culture is not consumed as the colony survival depends on its development, and during this period, the queen fertilizes it with fecal fluid13. Days after emerging, the first workers open the nest entrance and begin a foraging activity in the immediate area of the nest13. They incorporate the material collected as the substrate of the fungus garden, which is now serving as food for the workers13,22. Before being added to the fungus culture, the plant material carried in by the workers is cut into tiny pieces and moistened with fecal liquid13. The ants manipulate fungus inoculum to increase and control its growth, which will serve for partitioning big soil excavated chambers, specialized in conditioning the garden13,22,25.
About 6 months after the nuptial flight, A. sexdens nests contain a fungus chamber and a few channels. The great specialization in the construction of leaf-cutting ant nests works as a defense mechanism against natural enemies and unfavorable environmental factors22. Leaf-cutting ants are known to fragment the fungus garden and transpose it to chambers with high humidity when chambers start to dry out13. Thus, despite the excavation of the nest having a considerable energy cost, the energy invested is reversed in benefits for the colony itself22. With a few exceptions, Atta species also make specialized chambers for the colony&#39;s waste, made mostly of depleted fungus substrate and bodies of dead ants, isolating it from the rest of the nest, and establishing an important social immunity strategy30. In addition, a distinct group of workers manipulate the refuse directly, to avoid contamination of other individuals. Workers constantly forage to nurture the fungus, which is the main nutritional resource of the colony. However, they can feed on plant sap as well while cutting fragments. Plant material is carefully selected for the fungus garden maintenance and influenced by many factors such as leaf traits and properties of the ecosystem13.
The foraging strategy of leaf-cutting ants to obtain fresh material is highly complex, and combined with the high harvest demand of established colonies, result in considerable economic loss to agricultural producers and jeopardizes forest restoration areas22,31. Therefore, these ants can be categorized as pests in most areas where they may be encountered, ranging from southern United States to north-eastern Argentina11,13,22,32. The extinguishing of problematic colonies is challenging due to the series of adaptations inherent in the biology of these insects (i.e., social organization, foraging, fungus-cultivation, hygiene, and complex nest structures)33. Hence, the population control strategies are distinct from those generally applied to other insect pests, and mainly resort to attractive contaminated bait offerings33,34. However, as these ants can reject harmful substances for both the fungus and the colony individuals, and compromise cultivated fields33, new natural compounds and alternatives of control are constantly being tested33,35,36. As experiment results can hardly be monitored on field-tested colonies, preliminary essays are conducted in a controlled environment.
Thus, experimental protocols must be adapted to groups of interest considering the heterogeneous lifestyle of ants, supporting studies on a species level, and accounting for colonies as operational units, where one ant is an element of a complex superorganism11. The reports gathered until now concerning the Atta genus made it attainable to successfully collect and maintain colonies in laboratory conditions and acknowledge their basic needs and general functioning. Based on their natural processes such as reproduction, colony founding, and feeding behaviors, a routine of practices has been developed that permits the long-term establishment of colonies in different types of nests. Here, a procedural protocol to maintain leaf-cutting ants in the laboratory is described and highlights possible general research with distinct experimentation purposes and science outreach.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Entomologic forceps</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Glass tank</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Tempered glass, custom made</td>
		</tr>
		<tr>
			<td>Hose</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Transparent, PVC 1/2 Inch x 2,0 mm</td>
		</tr>
		<tr>
			<td>Latex gloves</td>
			<td>Descarpack</td>
			<td>550301</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Nitrile gloves</td>
			<td>Descarpack</td>
			<td>433301</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Open arena</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Polypropylene crate</td>
		</tr>
		<tr>
			<td>Plaster pouder</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Plaster pouder used in construction, must be absorbant</td>
		</tr>
		<tr>
			<td>Plastic Containers for collection</td>
			<td>Prafesta</td>
			<td>Natural C&#243;d.: 8231/Natural C&#243;d.: 8262</td>
			<td>Lidded, transparent , polypropylene</td>
		</tr>
		<tr>
			<td>Plastic containers for nests</td>
			<td>Prafesta</td>
			<td>Discontinued</td>
			<td>Polystyrene, hermetic</td>
		</tr>
		<tr>
			<td>Teflon</td>
			<td>Dupont</td>
			<td>N/A</td>
			<td>Polytetrafluoroethylene liquid (PTFE Dispertion 30)</td>
		</tr>
	</tbody>
</table>

collection and long-term maintenance of leaf-cutting ants (atta) in laboratory conditions

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled environments enables an enriched comprehension of their biology that can contribute to applied research. This practice is usually employed in population control studies of species that cause economic loss, such as Atta ants. To cultivate their mutualistic fungus, these leaf-cutting ants collect leaves and for this are considered agricultural pests widely distributed throughout the American continent. They are highly socially organized and inhabit elaborated underground nests composed of a variety of chambers. Their maintenance in a controlled environment depends on a daily routine of several procedures and frequent care that are described here. It starts with the collection of queens during the reproductive season (i.e., nuptial flight), which are then individually transferred to plastic containers. Due to the high mortality rate of queens, a second collection can be carried out about 6 months after the nuptial flight, when incipient nests with developed fungus wad are excavated, hand-picked, and placed in plastic containers. In the laboratory, leaves are daily provided to established colonies, and ant-produced waste is weekly removed along with remaining dry plant material. As the fungus garden keeps growing, colonies are transferred to different types of containers according to the experimental purpose. Leaf-cutting ant colonies are placed in interconnected containers, representing the organizational system with functional chambers built by those insects in nature. This setup is ideal to monitor factors such as waste amount, fungus garden health, and the behavior of workers and queen. Facilitated data collection and more detailed observations are considered the greatest advantage of keeping ant colonies in controlled conditions.

A flowchart depicting the process of ant collection is shown in Figure 6. Here, some results obtained employing the protocol of collection, maintenance, and nest setups described above are shown.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64154/64154fig6v2.jpg" /> 
Figure 6: Flowchart for collection of leaf-cutting ants&#39; colonies. Following the protoc...

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Collection and Long-Term Maintenance of Leaf-Cutting Ants Atta in Laboratory Conditions

Here, a protocol is described to successfully collect and maintain healthy Atta (Hymenoptera: Formicidae) ant colonies in laboratory conditions. Additionally, different nest types and configurations are detailed together with possible experimental procedures.

Collection and Long-Term Maintenance of Leaf-Cutting Ants (Atta) in Laboratory Conditions

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled ...

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JoVE Journal

Biology

2.4K Views.  São Paulo State University. Here, a protocol is described to successfully collect and maintain healthy Atta (Hymenoptera: Formicidae) ant colonies in laboratory conditions. Additionally, different nest types and configurations are detailed together with possible experimental procedures.

Video: Collection and Long-Term Maintenance of Leaf-Cutting Ants Atta in Laboratory Conditions

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

Długoterminowe Imaging komórkach ssaków przy dużym polu Mikroskopia

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account.
We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies.
We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. 
Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines.

We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because the larval body wall and most internal organs are transparent. However, continuous live imaging of intact Drosophila larvae for longer than 30 min has been challenging because it is difficult to noninvasively immobilizeimmobilizing larvae for a long time. Here we present a larval mounting method called LarvaSPA that allows for continuous imaging of live Drosophila larvae with high temporal and spatial resolution for longer than 10 hours. This method involves partially attaching larvae to the coverslip using a UV-reactive glue and additionally restraining larval movement using a polydimethylsiloxane (PDMS) block. This method is compatible with larvae at developmental stages from second instar to wandering third instar. We demonstrate applications of this method in studying dynamic processes of Drosophila somatosensory neurons, including dendrite growth and injury-induced dendrite degeneration. This method can also be applied to study many other cellular processes that happen near the larval body wall.

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

This protocol describes a method for mounting Drosophila larvae to achieve longer than 10 h of uninterrupted time-lapse imaging in intact live animals. This method can be used to image many biological processes close to the larval body wall.

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...